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Page 1: RAPID DETECTION OF GENETICALLY MODIFIED ORGANISMS … · RAPID DETECTION OF GENETICALLY MODIFIED ORGANISMS IN COTTON ... Four samples out of five were found to be GM positive with

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Int. J. LifeSc. Bt & Pharm. Res. 2012 Abhijit V Sahasrabudhe and Vishwanath S Khadye, 2012

RAPID DETECTION OF GENETICALLYMODIFIED ORGANISMS IN COTTON SEEDS

BY REAL TIME PCRVishwanath S Khadye1 and Abhijit V Sahasrabudhe1*

Research Paper

The ability to detect presence or absence of foreign gene(s) relies on high yields of pure DNAsamples. Cotton seeds contain polyphenols, polysaccharides and other secondary metaboliteswhich often interfere during DNA isolation. Here we present optimization of DNA isolation andReal Time PCR conditions for the detection of GMO in cotton seeds (Gossypium spp.). Themodified CTAB protocol includes addition of sodium bisulfite in combination with higher CTABconcentration and precipitation of DNA with 5 M NaCl in combination with chilled ethanol increasedthe solubility of polysaccharides and polyphenols. This protocol is devoid of use of expensivechemicals such as Proteinase K and RNase etc. We also optimized Real Time PCR conditionssuch as annealing temperature and concentration of DNA for the detection of CaMV 35S targetgene. Four samples out of five were found to be GM positive with reference to target gene.

Keywords: Cotton seeds, DNA isolation, GMO analysis, CaMV35S promoter, Real Time PCR

*Corresponding Author: Abhijit V Sahasrabudhe,[email protected]

INTRODUCTIONGenetically modified organisms (GMO’s) contain

specific traits/ genes which are inserted in to

organisms to improve their property which does

not generally occur by natural mating (Anklam et

al., 2002). Such genetically modified crops have

made a vital contribution to food, feed and fibre

security in today’s world. By the end of year 2010,

the global area of GM plants was 148 m ha which

was just 1.7 m ha in 1996. Now a day’s around

forty five GM crops are available of which soya,

cotton, maize and potato are major GM crops.

As far as India is concerned, GM cotton covers

ISSN 2250-3137 www.ijlbpr.comVol. 1, No. 4, October 2012

© 2012 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2012

1 DSPM’s K V Pendharkar College of Arts, Science and Commerce, MIDC, Dombivli (E), Thane, India.

around 10.0 m ha of land and is the only dominant

crop produced on commercial basis. BT cotton

was the first introduced by a USA based company

Monsanto in collaboration with Maharashtra

Hybrid Seed Company (MHYCO) and was

commercialized in 1990’s at the global level and

officially approved for sale in India in 2002. Cotton

is an important crop for textile industry and is the

food and feed source of many areas of the world.

It was developed to reduce heavy resilience on

pesticide; Bacillus thurengenesis naturally

produces a chemical harmful to a small fraction

to insects, larve of moths, butterflies, flies and

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Int. J. LifeSc. Bt & Pharm. Res. 2012 Abhijit V Sahasrabudhe and Vishwanath S Khadye, 2012

harmful to other forms of life. The gene coding

for BT toxin has been inserted in to cotton, causing

cotton to produce a natural insecticide in its

tissues. Cotton contains gossypol, a toxin that

makes it inedible. However scientists have

silenced the gene that produces a toxin thereby

making it a potential food crop.

In genetically modified crops most common

recombinant elements are Cauliflower Mosaic

Virus (CaMV 35S promoter) and NOS terminator

sequences (Lipp et al., 1999) both of which are

transcription regulating sequences (Odell et al.,

1985). As the demand to detect transgene is vastly

increasing due to introduction of new GM crops,

the development of rapid detection protocols have

also become much more challenging and

mandatory.

A series of analytical tools based on detecting

DNA, RNA and Protein are available to

differentiate between GM and non GM crops.

Among DNA based methods, Polymerase Chain

Reaction (PCR) is the most widely used method

in research as well as in commercial laboratories

because of its high sensitivity and specificity

(Anklam et al., 2002 and Ahmed, 2002). Real Time

PCR is preferred over conventional PCR as it

allows monitoring of reaction in real time through

fluorescence (Heid et al., 1996) plus no post

agarose gel detection is required in Real Time

PCR which used SYBR green dye I chemistry.

This has a high affinity towards all double stranded

DNA (Morrison et al., 1998) and a melting curve

is generated at the end of reaction allowing post

PCR identification in expected target as well as

in closely related targets. Another advantage is

that it is cost effective as no dye labeled

oligonucleotide probes is required (Terry and

Harris, 2001).

Cotton (Gossypium spp.) being a tree is a verydifficult plant for DNA isolation from leaves or fromseeds due to presence of phenolic compoundsthat generally interfere with quality and quantityof DNA by making it unstable for downstreamprocesses such as RFLP, RAPD and PCR etc.There are many protocols available for isolationof genomic DNA from cotton seeds, but all theseare laborious, time consuming and costly. Forcommercial basis a rapid and pure isolation ofgenomic DNA is a prerequisite. Taking this intoaccount, it was decided to standardize a rapidand cost effective method for isolation of genomicDNA from cotton seeds and optimizing Real TimePCR conditions for qualitative detection of target

gene (CaMV 35S promoter) in cotton seeds.

MATERIALS AND METHODSSample Collection

Five cotton seed samples were collected from

different regions of Maharashtra.

Chemicals and Other Reagents

Extraction buffer containing 2.5% CTAB, 1.8M

NaCl, 0.1M Tris-HCl (pH-8.0), 0.02M EDTA.2H20

(pH-8.2), 0.35 M Sodium bisulfite and 0.1%

-mercaptoethanol (added just before the use)

Phenol: Chloroform: isoamyl alcohol (25:24:1).

5M NaCl

Chilled distilled ethanol

0.1X TE buffer containing 10mM Tris (pH-8)

and 1 mM EDTA. 2H20 (pH-8)

Primer Information

Primers were synthesized from Sigma Aldrich,

USA. All primers were diluted to the working

concentrations 10pmol/l with sterile deionized

water. The sequences of the primers are given in

Table 1.

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Int. J. LifeSc. Bt & Pharm. Res. 2012 Abhijit V Sahasrabudhe and Vishwanath S Khadye, 2012

DNA Isolation

GMO CRL Protocol

1. Take 6 gms of cotton seed powder and add 25

ml of CTAB extraction buffer containing 0.5ml of

-mercaptoethanol and 0.25ml of Proteinase K.

Mix the content by inversion. 2. Incubate at 550C

for 60 mins with intermittent shaking. Cool the

tube after incubation. 3. Add 20 ml of Phenol:

Chloroform: Isoamylalchohol (25:24:1) and mix

gently by inversion. 4. Centrifuge at 13,000 g for

10 mins at room temperature. 5. Collect the

aqueous layer and repeat step 3 and 4 twice.

6. To the collected aqueous layer add 2 volumes

of chilled isopropanol and gently mix by inversion.

7. Allow the DNA threads to appear. 8 Spin the

DNA at 5000 rpm for 10 mins at 40C. 9. Remove

the supernatant and allow the pellet to dry

overnight 10. Next day dissolve the pellet in 2ml

of TE buffer and store at -200C. 11. Add 50µl of

RNase and incubate at 370C for 30 min. 12. To

extract DNA, add 4 ml of Chloroform:

Isoamylalchohol (24:1) and mix the content

vigorously. 13. Centrifuge at 10000 rpm for 10

min at room temperature. 14. To the upper layer,

add half volumes of 7.5M ammonium acetate and

add 2 volumes of 100% ethanol. 15. Allow the

DNA to appear again and spin the content at 40C

for 10 min at 5000 rpm. 16. Throw the supernatant

and allow the pellet to dry and next day add TE

buffer.

Standardized Protocol

1. Weigh 30 mg of finely grinded cotton seed

powder in a 2 ml microcentrifuge tube. 2. Add

250 µl of extraction buffer and mix well with gentle

inversion. 3. Incubate the content at 600C for one

hour with intermittent shaking after 10 min. 4.

Centrifuge at 12,000 rpm for 20 min at 250C. 5.

Collect the aqueous layer and add equal volume

of phenol: chloroform: isoamylalchol (25:24:1)

and mix well by gentle inversion. 6. Centrifuge at

12,000 rpm for 20 mins at 250C. 7. Repeat steps

5 and 6 twice. 8. To the aqueous layer, 0.5

volumes of 5 M NaCl was added followed by 2

volumes chilled distilled ethanol and mix by gentle

inversion. 9. Allow the DNA threads to appear. 10.

Spin the content at 40C for 10 min at 5000 rpm.

11. Throw the supernatant and allow the pellet to

dry overnight. 12. To the pellet, add 150 µl of 0.1X

TE buffer and store at –200C till use.

Agarose Gel Electrophoresis and DNAPurity

The genomic DNA was resolved on 0.8% agarose

gel in 1X TAE buffer with internal ethidium bromide

staining and electrophoresis was carried out at

65V for 2 hrs and visualized under UV

transilluminator. DNA purity was checked by

measuring the absorbance at 260 nm and 280

nm.

Real Time PCR

In order to check whether the extracted was

Table 1: Primers used for Real Time PCR study

Oligo Name Sequence GC%

CaMV forward GCTCCTACAAATGCCATCA 47%

CaMV reverse GATAGTGGGATTGTGCGTCA 50%

Sah 7 forward AGTTTGTAGTTTTGATGTTACATTGAG 32%

Sah 7 reverse GCATCTTTGAACCGCCTACTG 52%

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suitable for GMO detection, Real Time PCR was

performed using SYBR Green Master Mix. Real

Time PCR analysis was carried out in 7300 Real

Time PCR System (Applied Biosystems, USA).

All the amplification reactions were performed in

96 well plates. Individual reactions contains 12.5

µl of 2.5X SYBR Green Master mix, 5 µl of template

DNA, 1 µl of each forward and reverse primer

and finally volumised to 25 µl using sterile Mili-Q

water. Negative template control was also run

along with DNA samples to avoid the detection of

false positive and false negative. For positive

control we used standard cotton seed powder

DNA (0.1%) (ERM-BF413b) purchased from

European Union (EU) Joint Research Center,

Fluka. PCR conditions involved an initial

denaturation for 5 min at 950C, followed by 40

cycles of 950C for 15 sec, 600C for 1 min and

720C for 2 min. Finally reactions were held at 720C

for 5 min. The amplification products were

electrophoresed on 2% agarose gel for 1 hr at

100 volts with internal ethidium bromide staining

to confirm the desired product.

RESULTS AND DISCUSSIONIsolation of good quality of genomic DNA from

cotton seeds was very difficult due to presence

of phenolic compounds. During tissue

homogenization, these compounds oxidize and

thus irreversibly bind with proteins and other

nucleic acids. This irreversible binding produces

a gelatinous pellet with brownish color which is

hard to separate from organelles and thereby

making DNA unstable for downstream processes.

Most procedures for DNA isolation from plant

species containing high levels of phenolic

compounds are modified versions of standard

protocols. In our study, GMO-CRL method

reported earlier for isolation of genomic DNA from

modified cotton seeds did not work well. Though

we could able to isolate DNA but the pellet was

brownish having gel like appearance made it

difficult to dissolve in 0.1X TE buffer. Electro-

phoresis pattern showed presence of RNA along

with proteins and other impurities (Figure 1, lane

3 and 4). We also noticed that yield was very less

though starting material was around 3 gms.

A260/280

ratio was 2.4 with GMO-CRL protocol

indicating interference of RNA. Hence we have

devised a simpler procedure that used sodium

bisulfite as a reducing agent and extraction buffer

based on the CTAB method. We observed that

addition of 0.35 M sodium bisulfite to extraction

buffer drastically improved the DNA quantity and

quality, avoiding contamination and browning of

the pellet by phenolic compounds. The purity

decreased when we used lower concentration

(0.15 M) of sodium bisulfite and with higher

concentration (1M) the yield was less probably

because 1M sodium bisulfite fulfills a role of

osmoprotectant. In our case we just used 50 mg

of cotton seed powder as the starting material

which was very less as compared to GMO-CRL

method. During precipitation of DNA, we used

chilled ethanol which improved the yield. It has

been reported that higher concentrations of NaCl

along with ethanol increases the solubility of

polysaccharides and phenolic compounds (Fang

et al., 1992; Sahasrabudhe and Deodhar, 2010).

Thus we could able to isolate DNA free of RNA

and protein contamination (Figure 1, lane 2)

(depicted with black arrow). The freer the DNA is

from contaminants easier it is to resuspend the

pellet. Disadvantage in GMO-CRL protocol was

we need to re precipitate DNA again after RNase

treatment which makes it time consuming plus

there are chances of shearing of DNA on the other

hand, our standardized method is fast, reliable

and cost effective as no expensive chemicals

such as RNase and Proteinase K are required.

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Another advantage is that very less starting

material is required for isolation of DNA (DNA yield

was 1.015 µg with 30 mg of cotton seed powder.

Real Time PCR Analysis

Initially we tried amplification pattern of DNA

samples isolated from GMO-CRL protocol using

Real Time PCR. We observed quenching in the

amplification curve (Figure 2). The reason could

be because of inhibition of Taq DNA polymerase

due to the inhibitory components in the reaction

mixture, proteins, RNA or polyphenolics

contaminants present in the DNA sample. Also a

small amplification curve was seen in the target

gene of NTC. This might be because of the cross

contamination in the adjacent wells or may be

due to primer-dimmer formation. To overcome

these difficulties, we then standardized our DNA

extraction method to get pure and sufficient

amount of genomic DNA. Our standardized

method produced proper amplification curve

(Figure 3). After standardizing DNA isolation

protocol we then optimized various PCR cycle

parameters such as annealing temperature and

amount of DNA. We tried three annealing

temperatures such as 55, 60 and 650C. At 550C

there was a quenching seen in the amplification

curve while at 650C there was no amplification

but at 600C a proper curve was observed plus

there was no amplification in NTC (Non template

control) (Table 2, depicted with red arrow). For

Real Time PCR analysis, we used 20, 40 and 80

ng of DNA samples. We found that at 20 ng there

was no amplification curve but at 40 and 80 ng a

proper amplification curve seen. Hence for further

experiments we used 40 ng of DNA.

As mentioned earlier, we have tested five

different cotton seed samples collected from five

regions of Maharashtra. Interestingly among five

samples, four samples were found to be GM

positive since the amplification curve was

observed in target as well as in endogenous gene

(Figure 3). As we can see from the Table 2, Ct

Figure 1: Genomic DNA of Cotton Seeds Resolved on 0.8% Agarose Gel

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Int. J. LifeSc. Bt & Pharm. Res. 2012 Abhijit V Sahasrabudhe and Vishwanath S Khadye, 2012

Figure 2: Quenched Amplification Curve

values of four samples were earlier than that of

the standard GM (0.1%) target gene confirming

that there was early amplification of target gene

as compared to that of standard CaMV gene.

Only sample one showed undetermined Ct value

for target gene indicating complete absence of

CaMV promoter gene but a proper amplification

was observed in endogenous gene of the same

sample confirming it as GM negative (depicted

with black arrow). As the SYBR green dye I binds

non specifically to any double stranded DNA, the

measured florescence may have been

contributed by non specific PCR products or by

primer dimmers. In order to differentiate such

artifacts from specific PCR products, a melt

curve cycle was incorporated in to Real Time

Figure 3: Proper Amplification Curve

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PCR. The melting temperature (Tm) is usually

determined by products length, GC content,

concentration of dye and specific template in the

reaction tubes. Due to this non specific products

normally melt at much lower temperature than

that of specific products which are larger in size

(Taverniers et al., 2005). In our case, melting

temperature (Tm) of cotton one (Sample one)

where Ct value was undetermined, was 72 and

690C which was less than that of rest of the other

positive samples (Table 2).

After Real Time PCR cycle, agarose gel

electrophoresis was carried out for the confirmation

of deseired amplicons (Figure 4). It was observed

that in case of target gene, an amplicon of 107 bp

was prominent and that of endogeneous gene

product size was ~190 bp in all tested samples

confirming the proper Real Time PCR run.

Table 2: Table Showing Ct Values and Tm for Each Test Sample

Figure 4: PCR Product Resolved on 2% Agarose Gel Lane 1, 3 and 5: Endogenous Gene Product, 2, 4 & 6:

Target Gene Product and M: Molecular Marker

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CONCLUSIONPCR based methods for GMO detection appears

to be the method of choice because of its high

sensitivity and specificity. Genetic analysis of

plants relies on high yield of pure DNA sample

which is an essential prerequisite for the detection

of GMO’s in various crops. For this purpose we

initially standardized rapid DNA isolation protocol

devoid of expensive chemicals like RNase or

Proteinase K.

To conclude we can say that sodium bisulphite

CTAB based DNA isolation method and qualitative

Real Time PCR analysis is suitable for rapid

detection of GMO in cotton seeds on commercial

scale. It is possible to detect the presence of

transgene to an extent of 0.1%. As India will be

implementing mandatory labeling on GM crops

very soon, this qualitative approach will facilitate

the labeling legislations.

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